Evidence-based causation in toxicology: A 10-year retrospective.

We introduced Evidence-based Toxicology (EBT) in 2005 to address the disparities that exist between the various Weight-of-Evidence (WOE) methods typically applied in the regulatory hazard decision-making arena and urged toxicologists to adopt the evidence-based guidelines long-utilized in medicine (i.e., Evidence-Based Medicine or EBM). This review of the activities leading to the adoption of evidence-based methods and EBT during the last decade demonstrates how fundamental concepts that form EBT, such as the use of systematic reviews to capture and consider all available information, are improving toxicological evaluations performed by various groups and agencies. We reiterate how the EBT framework, a process that provides a method for performing human chemical causation analyses in an objective, transparent and reproducible manner, differs significantly from past and current regulatory WOE approaches. We also discuss why the uncertainties associated with regulatory WOE schemes lead to a definition of the term "risk" that contains unquantifiable uncertainties not present in this term as it is used in epidemiology and medicine. We believe this distinctly different meaning of "risk" should be clearly conveyed to those not familiar with this difference (e.g., the lay public), when theoretical/nomologic risks associated with chemical-induced toxicities are presented outside of regulatory and related scientific parlance.

Cyp3A regulation: from pharmacology to nuclear receptors.

Among the human liver cytochrome P450s (P450s), a family of microsomal hemoproteins responsible for catalyzing the oxidative metabolism of clinically used drugs and environmental chemicals, attention has been focused on CYP3A, a form that is the most abundant and is inducible by many of its substrates. From early pharmacological studies that demonstrated induction of CYP3A by glucocorticoids and, paradoxically, by antiglucocorticoids, the existence of a nonclassical glucocorticoid receptor mechanism was inferred and prompted research that culminated in the identification of a unique member of the nuclear receptor family, the pregnane X receptor (PXR; NR1I2). It has become increasingly evident that PXR as well as other nuclear receptors mediate CYP3A induction in a unique and complex manner including inducibility by structurally diverse compounds and striking interspecies differences in induction profiles. Future understanding of the role of nuclear receptors in regulating expression of CYP3A and other genes of the P450 family offers an exciting promise of further defining the physiologic function and interindividual differences of CYP3A in health and disease.

Reciprocal activation of xenobiotic response genes by nuclear receptors SXR/PXR and CAR.

The cytochrome P450 (CYP) gene products such as CYP3A and CYP2B are essential for the metabolism of steroid hormones and xenochemicals including prescription drugs. Nuclear receptor SXR/PXR (steroid and xenobiotic receptor/pregnenolone X receptor) has been shown both biochemically and genetically to activate CYP3A genes, while similar studies have established constitutive androstane receptor (CAR) as a CYP2B regulator. The response elements in these genes are also distinct, furthering the concept of independent regulation. Unexpectedly, we found that SXR can regulate CYP2B, both in cultured cells and in transgenic mice via adaptive recognition of the phenobarbital response element (PBRE). In a type of functional symmetry, orphan receptor CAR was also found to activate CYP3A through previously defined SXR/PXR response elements. These observations not only provide a rational explanation for the activation of multiple CYP gene classes by certain xenobiotics, but also reveal the existence of a metabolic safety net that confers a second layer of protection to the harmful effects of toxic compounds and at the same time increases the propensity for drug-drug interactions.

Identification by differential display of the IF1 inhibitor peptide of ATP synthase/ATPase as a gene inducible in rat liver by pregnenolone 16alpha-carbonitrile.

The synthetic steroid, pregnenolone-16alpha-carbonitrile (PCN), activates hepatic metabolism and elimination of xenobiotics mediated by its interaction with the PXR, a nuclear receptor that binds PCN and such glucocorticoids as dexamethasone (Dex). We used mRNA differential display to define further the domain of genes under the control of PCN/PXR. We found 76 cDNA fragments representing mRNAs differentially expressed in the liver of rats treated with PCN or Dex. Sequence analysis of one of these revealed a PCN induced cDNA fragment as IF1, an inhibitor peptide of ATP synthase/ATPase complex. Northern blot analysis revealed that IF1 was detectable in untreated liver and was induced 2-3 fold following treatment with PCN. IF1 mRNA was not detected in lung, heart, kidney, or testes of control or PCN treated rats. We conclude that IF1 inhibitor peptide is a novel representative of an apparently large set of previously unrecognized genes coordinately controlled by the PCN/PXR system to maintain homeostasis during toxic stress.

Humanized xenobiotic response in mice expressing nuclear receptor SXR.

The cytochrome CYP3A gene products, expressed in mammalian liver, are essential for the metabolism of lipophilic substrates, including endogenous steroid hormones and prescription drugs. CYP3A enzymes are extremely versatile and are inducible by many of their natural and xenobiotic substrates. Consequently, they form the molecular basis for many clinical drug-drug interactions. The induction of CYP3A enzymes is species-specific, and we have postulated that it involves one or more cellular factors, or receptor-like xeno-sensors. Here we identify one such factor unequivocally as the nuclear receptor pregnenolone X receptor (PXR) and its human homologue, steroid and xenobiotic receptor (SXR). We show that targeted disruption of the mouse PXR gene abolishes induction of CYP3A by prototypic inducers such as dexamethasone or pregnenolone-16alpha-carbonitrile. In transgenic mice, an activated form of SXR causes constitutive upregulation of CYP3A gene expression and enhanced protection against toxic xenobiotic compounds. Furthermore, we show that the species origin of the receptor, rather than the promoter structure of CYP3A genes, dictates the species-specific pattern of CYP3A inducibility. Thus, we can generate 'humanized' transgenic mice that are responsive to human-specific inducers such as the antibiotic rifampicin. We conclude that SXR/PXR genes encode the primary species-specific xeno-sensors that mediate the adaptive hepatic response, and may represent the critical biochemical mechanism of human xenoprotection.

Characterization of DNA-binding proteins required for glucocorticoid induction of CYP3A23.

Cytochrome P450 (CYP) 3A23 is transcriptionally regulated in rat liver by such glucocorticoids as dexamethasone (DEX) and by such antiglucocorticoids as pregnenolone 16 alpha-carbonitrile (PCN). Based on studies of CYP3A23 gene fragments expressed in primary cultures of adult rat hepatocytes and tested for DNA-protein interactions, we have proposed that the mechanism of CYP3A23 induction by these steroid hormones involves the glucocorticoid receptor or a protein induced by glucocorticoids indirectly interacting with proteins constitutively bound to an enhancer element consisting of a direct repeat of 7-bp separated by two nucleotides in the 5'-flanking region of the CYP3A23 gene (L. Quattrochi et al., J. Biol. Chem. 270, 28,917, 1995). In the present study, we prepared and transiently expressed in cultured rat hepatocytes 20-bp double-stranded (ds)-oligonucleotides containing this direct repeat or various mutations of this direct repeat inserted into a chloramphenicol acetyltransferase (CAT) reporter plasmid. We found that both repeats were necessary for induction of CAT by either DEX or PCN. Analysis of proteins bound to CYP3A23 enhancer through the use of uv cross-linking revealed two rat liver nuclear proteins with molecular masses of approximately 130 and 100 kDa, as well as several proteins of molecular masses between 45 and 60 kDa, that specifically bind to the 20-bp ds-oligonucleotide CYP3A23 enhancer. Methylation interference assays determined that all guanine residues within the direct repeats of this oligonucleotide are important for protein binding. Mutations of these guanine residues abolished binding of nuclear proteins and eliminated DEX or PCN inducibility of CAT. These data suggest that constitutively bound proteins, interacting with the CYP3A23 enhancer possibly as a heterodimeric complex, play a role in the glucocorticoid inducibility of CYP3A23.

Characterization of a pretranscriptional pathway for induction by phenobarbital of cytochrome P450 3A23 in primary cultures of adult rat hepatocytes.

Our laboratory has proposed that phenobarbital (PB), a typical lipophilic agent that induces some members of the supergene family of liver microsomal cytochromes P450 (e.g., CYP2B1/2 and CYP3A23), acts through a complex process inhibitable by the presence of growth hormone (GH), the absence of some components of the extracellular matrix, or a disrupted cytoskeleton. To verify that these manipulations of the culture environment block specific steps in the PB induction pathway rather than simply exerting nonspecific or toxic effects on CYP2B1/2 gene transcription, we have now examined PB induction of CYP3A23, a gene known to also be transcriptionally activated by dexamethasone (DEX) through a "nonclassical" pathway apparently involving the glucocorticoid receptor. We found that in primary cultures of adult rat hepatocytes treated with PB, induction of CYP3A23 mRNA, just as we reported for induction of CYP2B1/2 mRNA, required the use of Matrigel (a reconstituted basement membrane) and was blocked by the presence of cytoskeletal inhibitors (colchicine or cytochalasins) or of physiologic concentrations of GH in the culture medium. Moreover, PB induction of CYP3A23 and of CYP2B1/2 mRNAs was greatly diminished by inhibitors of cAMP-dependent protein kinase (PKA). In striking contrast, induction of CYP3A23 mRNA by DEX was unaffected by any of these alterations of the culture conditions that block its induction by PB. We conclude that the effects of extracellular matrix, GH, disruption of the cytoskeleton, and activation of cAMP-dependent protein kinase, pharmacologically define multiple, pretranscriptional steps in the pathway(s) for PB induction of liver cytochromes P450.

Relevance of rat liver tumors to human hepatic and endometrial cancer.

Concern for a risk of cancer in humans has been prompted by the appearance of hepatocellular carcinomas in tamoxifen-treated rats. However, there is no evidence of excess hepatocellular carcinoma among tamoxifen-treated women. Moreover, liver tumors are not induced in tamoxifen-treated mice or hamsters. An explanation for the species selectivity of this toxic effect may relate to the greater rate of formation of reactive intermediates in rats than either mice or humans. This results in persistent liver DNA adducts in tamoxifen-treated rat liver exceeding those produced in treated mice or in background levels measured in women taking tamoxifen. Another observation that reduces concern for human carcinogenesis is that bioactivation of tamoxifen may be inducible at dosages used in rodent cancer bioassays but not in those used clinically. Suggestions that endometrial cancer may be tamoxifen related are not supported by rodent data for endometrial cancer. Indeed, endometrial tissue lacks the necessary tamoxifen bioactivating capacity of liver consistent with the absence of DNA adducts in the endometrium of women taking tamoxifen. Finally, it seems doubtful that the colon is a target for human carcinogenesis of tamoxifen given the negative epidemiologic studies and high-dose rodent studies. In summary, there is at present no sound scientific basis for extrapolation of rat liver cancer findings to risks of liver, endometrial, and colon cancer in women receiving tamoxifen.

Mechanisms of alcohol impairment of recovery from mechanically denuded areas made within cultured rat hepatocytes.

The effect of ethanol on the ability of a denuded hepatocyte cell surface to recover is unknown. We therefore determined the effect of ethanol on the rate of renewal of mechanical wounds made in near-confluent monolayers of primary cultures of rat hepatocytes. We found that ethanol exerted a dose-dependent effect to impair rat hepatocyte recovery and that at least 12 hours of exposure to ethanol was required to induce this impairment. The effect of ethanol to impair recovery of denuded areas of epithelium was not seen in two established renal tubular epithelial cell lines. Ethanol impairment of rat hepatocyte recovery could be blocked by an alcohol dehydrogenase inhibitor (4-methyl pyrazole) and potentiated by an inhibitor of aldehyde dehydrogenase (pargyline). The effect of ethanol to inhibit rat hepatocyte recovery of denuded areas was not associated with any change in hepatocyte cell surface expression of the beta1 integrin subunit as determined by flow cytometry. These results suggest that acetaldehyde is responsible for ethanol inhibition of hepatocyte recovery from mechanical injury and that this impairment occurs independent of cell surface expression of the beta1 integrin subunit.

Mechanisms of recovery from mechanical injury of cultured rat hepatocytes.

The mechanism(s) whereby hepatocytes restore denuded areas remains unknown. We therefore studied the recovery of denuded areas made in monolayers of primary cultures of rat hepatocytes. Minimal recovery occurred in cells plated on plastic. Plating on Matrigel produced modest recovery (25% at 24 h), whereas plating on a type I collagen substrate resulted in > 70% recovery at 24 h. The rate of recovery on collagen could be attenuated by a monoclonal antibody directed against the extracellular domain of the beta 1-integrin subunit. Monoclonal antibodies directed against CD44 (the hyaluron receptor) and E-cadherin did not influence the rate of recovery. Recovery could be stimulated, in a dose-dependent fashion, by epidermal and hepatocyte growth factors. The effects of epidermal and hepatocyte growth factors to promote recovery occurred in the absence of 5-bromo-2'-deoxyuridine uptake, suggesting a proliferation-independent mechanism. Transforming growth factor-beta 1 inhibited recovery. Exposure to selected cytokines (interleukins 1 and 2), an adenine nucleotide [adenosine 5'-O-(3-thiotriphosphate)], adenosine, pertussis toxin, and selected agents that bind to fibronectin and other matrix component adhesive sites (heparin and the RGD peptide) did not influence the rate of recovery of hepatocytes. However, the peptide DGEA, which can bind to collagen adhesive sites, attenuated recovery. These studies demonstrate that primary cultures of rat hepatocytes require a particular type of extracellular matrix to renew denuded areas and that the beta 1-integrin subunit may be involved in this recovery process. Hepatocyte recovery of denuded areas can be modulated by growth factors in both a stimulatory (epidermal and hepatocyte growth factors) and an inhibitory (transforming growth factor-beta 1) fashion.

Trans-species gene transfer for analysis of glucocorticoid-inducible transcriptional activation of transiently expressed human CYP3A4 and rabbit CYP3A6 in primary cultures of adult rat and rabbit hepatocytes.

Interindividual variation in the spontaneous and in the glucocorticoid-or rifampicin-inducible expression of the CYP3A cytochromes P450, the dominant froms of this supergene family that catalyze the oxidation of numerous drugs and environmental chemicals in human liver, remains largely unexplained, due in part to the lack of a validated animal model. We analyzed the 5'-flanking sequences of CYP3A genes from the rat (CYP3A23, CYP3A2), rabbit (CYP3A6), and human (CYP3A4, CYP3A5, CYP3A7) and found variable regions separated by three areas (consensus I, II, and III) of sequence homology immediately upstream of their respective promoters. We used trans-species gene transfer in cellulo as a new approach for determining the basis for qualitative differences among species in liver expression of different forms of CYP3A. When we transfected into cultured rat hepatocytes vectors containing 5'-flanking DNA from CYP3A23, CYP3A4, or CYP3A6 genes, we found that CAT activity was induced on treatment with dexamethasone or pregnenolone-16 alpha-carbonitrile only if consensus II sequences were included. Rifampicin treatment had no effect. When the same constructions containing consensus II were transfected into rabbit hepatocytes, increased activity was observed on treatment of the cells with dexamethasone or with rifampicin but not with pregnenolone-16 alpha-carbonitrile. These results suggest that the host cellular environment rather than the structure of the gene dictates the pattern of CYP3A inducibility. The application of this new model system will provide a unique technique for identifying mechanisms of induction and advancing the development of appropriate toxicological models for human safety assessment.

Inhibitory actions of cyclic adenosine monophosphate and pertussis toxin define two distinct epidermal growth factor-regulated pathways leading to activation of mitogen-activated protein kinase in rat hepatocytes.

Increased intracellular cyclic adenosine monophosphate (cAMP) levels have been shown in some reports to inhibit and in other studies to stimulate growth factor-mediated activation of the mitogen-activated protein kinase (MAP kinase) pathway, depending on the cell type examined. The relationship between cAMP and MAP kinase in hepatocytes has not been examined. In the current study, stimulation of primary cultures of rat hepatocytes with hepatocyte growth factor (HGF) or epidermal growth factor (EGF) increased Ras, Raf, and MAP kinase activity. Incubation of hepatocytes with cAMP-increasing agents blocked activation of Raf by both HGF and EGF, whereas activation of Ras was unaffected. MAP kinase activation by HGF was completely inhibited, whereas EGF-stimulated MAP kinase activity was only slightly reduced. Incubation of hepatocytes with pertussis toxin slightly blunted MAP kinase activation by EGF but not HGF. Increasing cAMP in hepatocytes preincubated with pertussis toxin completely inhibited the activation of MAP kinase by EGF. In conclusion, HGF activates MAP kinase in hepatocytes exclusively through an Raf-dependent pathway and this activation may be completely blocked by increasing cAMP. In contrast, EGF activates MAP kinase in hepatocytes through both Raf-dependent and Raf-independent pathways: the latter pathway probably involves a pertussis toxin-sensitive G protein.

Identification of a novel dexamethasone responsive enhancer in the human CYP3A5 gene and its activation in human and rat liver cells.

The human liver cytochromes P450 3A (CYP3As), orthologous to the rat glucocorticoid inducible forms, are composed of at least four differentially expressed members. To begin the study of the molecular events in the glucocorticoid regulation of CYP3A5, we fused 5' sequences of CYP3A5 to the chloramphenicol acetyltransferase gene in a vector that contains the herpes simplex virus thymidine kinase promoter. In HepG2 cells, the largest 5' CYP3A5 gene fragment (1.4 kb) suppressed the TK promoter. However, suppression was overcome by addition of 10 microM dexamethasone. A series of unidirectional deletions revealed a unique 219-bp fragment (-891 to -1109 bp upstream from the transcriptional start site) that conferred dexamethasone responsiveness on the TK promoter regardless of either the distance or orientation from the promoter and thus appears to be an enhancer. Nucleotide sequence analysis of this CYP3A5 enhancer revealed no consensus 15-bp glucocorticoid responsive element (GRE) (GGTACANNNTGTTCT); however, two GRE "half-sites" (TGTTCT) were found separated by 160 bp. Although dexamethasone stimulated the CYP3A5 enhancer only 3-4-fold in HepG2 cells, the CYP3A5 enhancer was stimulated 7- and 12-fold in immortalized primary human hepatocytes and primary rat hepatocyte cultures, respectively. The glucocorticoid receptor (GCR) seems to be indispensable to this process because 1) dexamethasone induction can be blocked by the antiglucocorticoid RU-486, 2) dexamethasone-dependent transcriptional activation of the CYP3A5 enhancer in HepG2 cells required cotransfection of an expression vector containing the intact GCR, yet 3) cotransfection with a plasmid that contains a mutation in the ligand binding domain of the GCR does not activate the CYP3A5 enhancer in the presence of dexamethasone. To further localize the dexamethasone responsive region of the 219-bp CYP3A5 enhancer, it was subdivided and fused to the TKCAT expression vector. Transfection analysis in HepG2 cells demonstrated that neither GRE half-site can independently confer dexamethasone responsiveness on the TK promoter. Block mutations of either of the two GRE half-sites or point mutations at specific GCR binding sites eliminates dexamethasone inducibility, demonstrating the half-sites need to interact. Electromobility shift assays indicate that the CYP3A5 5'-GRE half-site 1) specifically binds purified GCR, 2) can displace binding of the GCR to a consensus GRE, and 3) shifts a protein in HepG2 nuclear extracts that is supershifted by GCR antibody, demonstrating that this enhancer is an authentic GRE. This is the first study to demonstrate that a member of the human CYP3A gene family contains an enhancer that binds the GCR and that this binding is critical to transcriptional activation by dexamethasone.

A novel cis-acting element in a liver cytochrome P450 3A gene confers synergistic induction by glucocorticoids plus antiglucocorticoids.

The induction by dexamethasone of rat liver CYP3A1 differs from classical glucocorticoid gene regulation in part because both glucocorticoids and antiglucocorticoids such as pregnenolone 16 alpha-carbonitrile (PCN) induce CYP3A1 through transcriptional gene activation. In the present study, we transiently expressed in primary cultures of rat hepatocytes plasmids consisting of CYP3A1 5'-flanking sequences fused to a chloramphenicol acetyltransferase reporter plasmid. Deletional analysis identified a 78-base pair (bp) element located approximately 135 bp upstream of the transcriptional start site that was inducible by treatment of the cultures with dexamethasone or PCN and was induced synergistically by dexamethasone plus PCN. Nuclear extract from control rat liver protected two regions within the 78-bp sequence against digestion with DNase I. The same two regions were protected when nuclear extracts from dexamethasone-treated animals were used. Analysis of both of the "footprints" (FP1 and FP2) failed to reveal a classical sequence for the glucocorticoid-responsive element. A 33-bp element that includes FP1 sequences inserted into the chloramphenicol acetyltransferase reporter plasmid and transiently expressed in rat hepatocytes conferred a profile of dexamethasone and PCN induction similar to that of the 78-bp element. However, an Escherichia coli expressed glucocorticoid receptor protein failed to protect sequences within FP1 in DNase I footprinting experiments and failed to change its mobility in gel shift assays. Moreover, as judged by the gel shift assay, the specific protein binding to this fragment was the same whether nuclear extracts from the liver of untreated or dexamethasone-treated rats were used. We conclude that the activation of CYP3A1 gene transcription by glucocorticoids may involve proteins already bound to the controlling element in the CYP3A1 gene through a mechanism in which GR in the presence of hormone does not bind directly to CYP3A1 DNA.

Critical role of extracellular matrix on induction by phenobarbital of cytochrome P450 2B1/2 in primary cultures of adult rat hepatocytes.

BACKGROUND: Although it has been known for more than three decades that administration of lipophilic chemicals, including phenobarbital, produces liver hypertrophy, proliferation of smooth endoplasmic reticulum, and induction of liver microsomal enzymes such as cytochromes P450 (CYP) 2B1 and 2B2, the mechanism of this adaptive response remains largely unknown. An important advance was the recognition that, unlike cultures of continuously proliferating liver cell lines or cultures of primary non-proliferating adult rat hepatocytes maintained on either plastic or collagen-coated dishes, hepatocytes cultured on a basement membrane gel, Matrigel, formed rounded clusters and permitted phenobarbital-mediated induction in vitro of CYP 2B1/2 mRNAs and immunoreactive proteins (1). EXPERIMENTAL DESIGN AND RESULTS: We cultured adult rat hepatocytes on Type 1 collagen (Vitrogen) and allowed the cells to spread, flatten, and firmly attach to the substratum. Subsequent incubation in medium containing Matrigel as a soluble component, fully restored, in a dose-dependent manner, the ability to respond to phenobarbital with induction of CYP 2B1/2 mRNAs. Repeating this experiment with medium containing equivalent amounts of purified laminin, a major component of Matrigel, or with YIGSR or SIKVAV, two peptides known to mimic various activities of laminin, similarly restored phenobarbital responsiveness to hepatocytes cultured on Vitrogen. In contrast, use of equal amounts of SHA-23, a scrambled peptide relevant to SIKVAV, produced no such effect. None of these treatments caused a rounding or any other observable change in the flattened, cellular morphology, making it unlikely that cell-spreading or alterations in cell shape account for loss of such differentiated liver functions as phenobarbital induction of CYP 2B1/2 mRNAs in cultured hepatocytes on Vitrogen. Hepatocytes cultured on Matrigel in the presence of either colchicine, cytochalasins B and D, nocodazole, or taxol did not show induction of 2B1/2 mRNAs by phenobarbital specifically, while the amounts of both albumin and glucose-6-phosphate dehydrogenase (G6PD) mRNAs were unaffected. CONCLUSIONS: We conclude that the process by which phenobarbital induced 2B1/2 mRNAs in hepatocytes appears to require highly concerted effects of specific extracellular components prominently involving laminin. This likely occurs through a signal transduction process requiring probably both microfilament and microtubular integrity.

Tyrosine kinase growth factor receptors but not seven-membrane-spanning receptors or phorbol esters activate mitogen-activated protein kinase in rat hepatocytes.

The response of rat hepatocytes to hormones and growth factors has been extensively studied with respect to phospholipase regulation and calcium mobilization. However, the mitogen-activated protein (MAP) kinase cascade which integrates signals from a wide variety of extracellular stimuli has not been examined in these cells. Thus, in the present study the pathways leading to activation of MAP kinase in primary cultures of adult rat hepatocytes were investigated. Growth factors acting through tyrosine kinase receptors (epidermal growth factor and hepatocyte growth factor) increased Raf and MAP kinase activity through a protein kinase C and calcium-independent pathway. Agonists acting through seven-membrane-spanning receptors (arginine vasopressin and angiotensin II) increased intracellular calcium concentration but did not stimulate Raf or MAP kinase activity. Arginine vasopressin, however, stimulated MAP kinase activity in rat 1a fibroblasts transfected with the hepatic V1a receptor and in rat aortic vascular smooth muscle cells. Phorbol 12-myristate 13-acetate (PMA) was also unable to stimulate Raf and MAP kinase in hepatocytes in spite of a marked activation of protein kinase C. We conclude that only signals arising from tyrosine kinase receptors are able to activate MAP kinase in hepatocytes. Neither agonists acting through seven-membrane-spanning receptors nor phorbol esters stimulate MAP kinase in hepatocytes. The results suggest that specific cellular components that link seven-membrane-spanning receptors with MAP kinase activation in tissues such as vascular smooth muscle are absent in rat hepatocytes.

Comparative analysis of cytochrome P4503A induction in primary cultures of rat, rabbit, and human hepatocytes.

We previously demonstrated that induction of hepatic cytochrome P4503A (CYP3A) immunoreactive protein is a response in rats, but not rabbits, treated with the antiglucocorticoid, pregnenolone 16 alpha-carbonitrile and in rabbits, but not rats, treated with rifampicin. These striking interspecies differences in response to CYP3A inducers prompted us to compare the effects of a variety of agents on CYP3A expression in primary cultures of hepatocytes from rats, rabbits, and humans, maintained under nearly identified conditions on Matrigel. We used treatment with dexamethasone, the most effective inducer of CYP3A mRNA and CYP3A immunoreactive protein in cultures of rat hepatocytes, to define the 100% response. As expected from their effects in vivo, incubations of cultures with medium containing pregnenolone 16 alpha-carbonitrile or rifampicin induced CYP3A mRNA to high levels exclusively in rat hepatocytes or rabbit hepatocytes, respectively. Pregnenolone 16 alpha-carbonitrile treatment also did not induce CYP3A immunoreactive protein in rabbit hepatocytes, although rifampicin treatment did increase CYP3A immunoreactive protein levels in rat hepatocyte cultures. Additions of phenobarbital to the cultures induced CYP3A mRNA and CYP3A immunoreactive protein to a greater extent in rabbit hepatocytes (94-108% of the dexamethasone response) than in rat hepatocytes (38-57% of the dexamethasone response). In primary cultures of human hepatocytes, dexamethasone and phenobarbital treatments induced CYP3A mRNA (> or = 4.4- and 1.9-fold, respectively, over the amounts of CYP3A mRNA in incubated control cultures).(ABSTRACT TRUNCATED AT 250 WORDS)